The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA2 in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA2 level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β0-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.
Materials and Methods
β-thalassemia is a genetic disorder resulting in decreased (β+-thalassemia) or completely absent (β0-thalassemia) production of the normal β-globin chain. The common β0-thalassemias in Southeast Asia are the codon 17(A>T), 41/42(–TCTT), and 71/72(+A) mutations and 3.5-kb deletion.1,2 People who have inherited 2 β-thalassemia genes have thalassemia major.3,4 Genetic counseling, carrier screening, and antenatal diagnosis programs have become more widespread and highly effective in reducing the incidence of β-thalassemia major.5–7 Selective antenatal analysis for β-thalassemia trait generally relies on RBC indices (mean corpuscular volume [MCV], mean corpuscular Hb, mean corpuscular Hb concentration, and RBC distribution width), the 1-tube osmotic fragility test, and the HbA2 value.8–11 Women with HbA2 values from 4% to 9.9% are diagnosed as having β-thalassemia. The women are referred to genetic counseling and testing for the spouse.
Today, the state-of-art method for quantification of HbA2 is high-performance liquid chromatography (HPLC),12,13 whereas capillary electrophoresis (CE) is a possible alternative high-throughput analysis method.14 Although the quantification of HbA2 by HPLC and CE is precise, there are some limitations. The falsely increased HbA2 values, which were higher than 4%, were observed in some patients with HbS when samples were analyzed by using HPLC. They were also found in some patients with HbE and HbC when samples were analyzed by using the CE method.15 However, there are no reports about the increased HbA2 values in patients with Hb Hope [β136(H14)Gly → Asp, GGT>GAT], the unstable Hb variant of the β-globin chain that is frequently found in the Thai population.16 Thus, the aim of this study was to compare the values of HbA2 in patients with Hb Hope analyzed by HPLC and CE.
The EDTA blood samples submitted to the laboratory of the Associated Medical Sciences Clinical Service Center, Chiang-Mai, Thailand, for hemoglobinopathy and thalassemia diagnosis were used in this study. Hemoglobin, hematocrit, and MCV values of RBCs were estimated with an automated blood counter (Sysmex KX-21, Sysmex, Kobe, Japan). The β-thalassemia and hemoglobinopathies were analyzed using HPLC (VARIANT β-thalassemia Short Program, Bio-Rad Laboratories, Hercules, CA). Blood samples with Hb Hope were further analyzed by the CE method (CAPILLARYS 2, Sebia, Norcross, GA) within 24 hours.
Genomic DNA was extracted from blood samples containing Hb Hope by using the NucleoSpin kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. The DNA was stored at −20°C until used. The β0-thalassemia codon 17, 41/42, and 71/72 mutations were identified by the multiplex amplification refractory mutation system polymerase chain reaction with SYTO9 and high-resolution melting (HRM) analysis according to the protocol described previously.1 The β0-thalassemia 3.5-kb gene deletion and α-thalassemia-1 Southeast Asian–type deletion were also identified in the samples by real-time polymerase chain reaction with SYBR Green1 (QuantiFast SYBR Green PCR, QIAGEN, Hilden, Germany) and HRM analysis as previously described.17
Blood Samples and Hemoglobin Analysis
The levels of Hb Hope and HbA2 measured by HPLC and CE in the same sample were compared by using the t test. P values less than .05 were considered to be statistically significant.
DNA Preparation and Molecular Analysis for β0-Thalassemia Mutations
A peak of Hb Hope was found in 11 blood samples when analyzed by HPLC and/or CE. Examples of HPLC chromatogram and CE electropherogram of Hb analyses from blood samples containing Hb Hope are shown in Figure 1. The specific peaks of Hb Hope and HbA2 analyzed by HPLC were observed at the retention times of 1.3 to 1.4 and 3.5 to 3.6 minutes, whereas those analyzed by CE were observed at zones 10 and 3, respectively.
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The Hb, MCV, and molecular analysis results for 11 blood samples are given in Table 1. The mean percentage of Hb Hope measured by CE was not statistically significantly different compared with that measured by HPLC (mean ± SD, 42.85% ± 7.09% vs 46.37% ± 4.21%; P = .21). It is interesting that the percentage of HbA2 measured by CE in all 11 samples was equal or higher than 4% and it was significantly higher than measured by HPLC (mean ± SD, 4.68% ± 0.60% vs 3.35% ± 0.84%; P = .009). Mean ± SD values of Hb, hematocrit, and MCV were observed at 12.61 ± 2.60 g/dL (126.1 ± 26.0 g/L), 38.25% ± 7.28% (0.38 ± 0.07), and 78.64 ± 7.65 μm3 (78.64 ± 7.65 fL), respectively. The α-thalssemia-1 SEA-type deletion was found in 1 sample with an MCV of 62 μm3 (62 fL). All DNA samples showed negative results for the analysis of β0-thalassemia codon 17, 41/42, and 71/72 mutations and β0-thalassemia 3.5-kb deletion (Table 1).
Supported in part by grants from the National Research Council of Thailand, Bangkok.
In the present study, SYTO9 and SYBR GREEN1 with HRM analysis were applied for the diagnosis of β0-thalassemia codon 17, 41/42, and 71/72 mutations and the β-thalassemia 3.5-kb deletion. Based on this approach, the most frequent (70%-90%) β0-thalassemia mutations found in the Southeast Asian population can be characterized. The heterozygous Hb Hope is usually associated with an asymptomatic clinical situation, but when this Hb is associated with β-thalassemia, the clinical expression could be anemia.20 Although the anemia indicated by the decrease of Hb and hematocrit values was found in some samples, β0-thalassemia mutations were not found in all 11 samples. This result suggested that the elevated HbA2 measured by CE may be due to the coelution of several Hb Hope adducts with HbA2, not because of β-thalassemia.
The HPLC peaks at the retention time of 1.4 minutes or at zone 10 of CE might represent the peak of Hb New York [β113(G15)Val → Glu, GTG>GAG], Hb Pyrgos [β83(EF7) Gly → Asp, GGC>GAC], and Hb Kodaira [β146(HC3)His → Gln, CAC>CAA], all of which are the other β-chain Hb variants and may affect the β-thalassemia diagnosis. In the Thai population, the incidence of Hb Hope is higher than the other aforementioned variants. Molecular analysis achieved by direct DNA sequencing showed that all 11 samples are Hb Hope. Therefore, the HPLC peak at the retention time of 1.4 minutes or at zone 10 of CE is the peak of Hb Hope in this case.
An elevated HbA2 level in samples containing Hb Hope measured by CE leads to an incorrect diagnosis of coinheritance of β-thalassemia with Hb Hope. Thus, the results of the RBC indices (MCV), the osmotic fragility test, and molecular analysis should be considered as they provide great value for β-thalassemia investigations.
We thank technicians in the Associated Medical Sciences Clinical Service Center, Associated Medical Sciences, Chiang-Mai University, for help and assistance. We are grateful to Gerald W. Rock and Kallayanee Treesuwan for editing the manuscript.